The vector sequence has been compiled using the information from sequence databases, published literature, and other sources, together with partial sequences obtained by Evrogen. This vector has not been completely sequenced.
pmKate2-peroxi is a mammalian expression vector intended for far-red fluorescent labeling of peroxisomes in living cells. The vector encodes far-red fluorescent protein mKate2 (see reporter description) targeted to the matrix of peroxisomes by tripeptide SKL (peroxisomal targeting signal, PTS) fused to the mKate2 C-terminus.
Transiently transfected HeLa cells expressing mKate2 fusion with peroxisomal targeting signal.
Scale bar represents 10 μm. Image from Shcherbo et al., 2009.
mKate2 codon usage is optimized for high expression in mammalian cells (humanized) [Haas et al., 1996]. To increase mRNA translation efficiency, Kozak consensus translation initiation site is generated upstream of the mKate2 coding sequence [Kozak, 1987].
pmKate2-peroxi vector can be used as a source of mKate2-PTS hybrid sequence. The vector backbone contains unique restriction sites that permit its excision and further insertion into expression vector of choice.
Note: The plasmid DNA was isolated from dam+-methylated
The vector backbone contains immediate early promoter of cytomegalovirus (PCMV IE) for protein expression, SV40 origin for replication in mammalian cells expressing SV40 T-antigen, pUC origin of replication for propagation in
SV40 early promoter (PSV40) provides neomycin resistance gene (Neor) expression to select stably transfected eukaryotic cells using G418. Bacterial promoter (P) provides kanamycin resistance gene expression (Kanr) in
Expression in mammalian cells
pmKate2-peroxi vector can be transfected into mammalian cells by any known transfection method. CMV promoter provides strong, constitutive expression of the mKate2-PTS fusion in eukaryotic cells. If required, stable transformants can be selected using G418 [Gorman, 1985].
Suitable host strains for propagation in
Location of features
PCMV IE: 1-589
Enhancer region: 59-465
TATA box: 554-560
Transcription start point: 583
Start codon (ATG): 679-681
Start of mKate2 coding sequence (ATG): 679-681
Peroxisomal Targeting Signal 1 (PTS1): 1387-1395
Stop codon: 1396-1398
SV40 early mRNA polyadenylation signal
Polyadenylation signals: 1550-1555 & 1579-1584
mRNA 3' ends: 1588 & 1600
f1 single-strand DNA origin: 1647-2102
Bacterial promoter for expression of Kanr gene
-35 region: 2164-2169
-10 region: 2187-2192
Transcription start point: 2199
SV40 origin of replication: 2443-2578
SV40 early promoter
Enhancer (72-bp tandem repeats): 2276-2347 & 2348-2419
21-bp repeats: 2423-2443, 2444-2464 & 2466-2486
Early promoter element: 2499-2505
Major transcription start points: 2495, 2533, 2539 & 2544
Kanamycin/neomycin resistance gene
Neomycin phosphotransferase coding sequences:
Start codon (ATG): 2627-2629
Stop codon: 3419-3421
G->A mutation to remove Pst I site: 2809
C->A (Arg to Ser) mutation to remove BssH II site: 3155
Herpes simplex virus (HSV) thymidine kinase (TK) polyadenylation signal
Polyadenylation signals: 3657-3662 & 3670-3675
pUC plasmid replication origin: 4006-4649
mKate2-related materials (also referred to as "Products") are intended for research use only. The Products are covered by U.S. Pat. 7,638,615; European Pat. 1994149; and other Evrogen Patents and/or Patent applications pending. By use of these Products, you accept the terms and conditions of the applicable Limited Use Label License.
The CMV promoter is covered under U.S. Patents 5,168,062 and 5,385,839, and its use is permitted for research purposes only. Any other use of the CMV promoter requires a license from the University of Iowa Research Foundation, 214 Technology Innovation Center, Iowa City, IA 52242.
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